Bone morphogenetic protein 15 and growth differentiation factor 9 co-operate to regulate granulosa cell function

Abstract

The oocyte-secreted polypeptide growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15, also known as GDF9B) have both been shown to be essential for ovarian follicular growth and function. The effects of murine (m) and ovine (o) GDF9 as well as oBMP15, alone or together, on 3 H-thymidine uptake and progesterone and inhibin production by granulosa cells from rats were determined. Murine GDF9 stimulated thymidine incorporation by granulosa cells whereas oGDF9 and oBMP15 alone had no effect. However, oBMP15 given together with mGDF9 or oGDF9 was very potent in stimulating 3 H-thymidine incorporation by granulosa cells with a greater than 3-fold stimulation compared with any growth factor alone. The synergistic effect of oBMP15 and oGDF9 was almost completely blocked by antibodies generated against these growth factors when administered either alone or in combination. While neither GDF9 (murine or ovine) nor oBMP15 were able to modulate FSH-stimulated progesterone production on their own, FSH-stimulated progesterone production by granulosa cells was potently inhibited when BMP15 and GDF9 were administered together. Immunoreactive ainhibin levels increased more than 15-fold from granulosa cells when BMP15 and GDF9 were given together whereas consistent stimulatory effects of either growth factor alone were not observed. The effects of GDF9 and BMP15, when added together, were different than those observed for the growth factors alone. Therefore, we hypothesize that within the ovary, these oocyte-secreted growth factors co-operate to regulate proliferation and gonadotropin-induced differentiation of granulosa cells in mammals.Reproduction (

Bone morphogenetic protein 15 and growth differentiation factor 9 co-operate to regulate granulosa cell function in ruminants

“…This is consistent with the finding that hBMP15 stimulates thymidine incorporation in rat and human granulosa cells (Otsuka et al 2000, Di Pasquale et al 2004. However, using the same batch preparation as described herein, oBMP15 did not stimulate 3 H-thymidine incorporation in rat granulosa cells (McNatty et al 2005). These data are also consistent with the previously stated hypothesis for GDF9, that the functions of the BMP15 may vary between species of origin of granulosa cells.…”

“…Indeed, in the present study, mGDF9 caused a small, but significant, suppression of thymidine incorporation in both ovine and bovine granulosa cells. This is in contrast to the stimulatory effect of mGDF9 on 3 H-thymidine incorporation in rodent granulosa cells (Vitt et al 2000, McNatty et al 2005. This finding is consistent with the hypothesis that the function of GDF9 may vary between the species of origin of granulosa cells (Shimasaki et al 2004).…”

“…The mGDF9, oGDF9 and oBMP15 constructs used for generating stably transfected cell lines has been described previously , McNatty et al 2005. The recombinant protein was produced into serum-free harvesting medium (DMEM/Ham's F-12, 1:1) supplemented with L-glutamine and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin), 0.01% (w/v) BSA (Sigma, Auckland, NZ) and 100 mg/ml heparin (Sigma, Auckland, NZ).…”

“…The recombinant protein was produced into serum-free harvesting medium (DMEM/Ham's F-12, 1:1) supplemented with L-glutamine and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin), 0.01% (w/v) BSA (Sigma, Auckland, NZ) and 100 mg/ml heparin (Sigma, Auckland, NZ). The amounts of GDF9 or BMP15 in conditioned media were determined by Western blotting as described previously (McNatty et al 2005). The reference standards used to assess the concentrations of mammalian-derived GDF9 and BMP15 were E. coli produced oGDF9 and oBMP15.…”

“…These observations could be related to differences in responses of granulosa cells to GDF9 and BMP15 or, potentially, to divergent affects of the growth factors from different species. We have recently shown that GDF9 and BMP15 co-operate in vitro to modulate functions of granulosa cells from rats (McNatty et al 2005). However, little is known about the roles of these growth factors and their potential interactions in ruminants.…”